Antisense technologies are a suite of techniques that, together, form a very powerful weapon for studying gene function (functional genomics) and for discovering new and more specific treatments of diseases in humans, animals, and plants (antisense therapeutics). A conventional definition of antisense refers to the laboratory manipulation and/or modification of DNA or RNA so that its components (nucleotides) form a complementary copy of normal, or “sense,” messenger RNA (mRNA). The binding, or hybridization, of antisense nucleic acid sequences to a specific mRNA target will, through a number of different mechanisms, interrupt normal cellular processing of the genetic message of a gene. This interruption, sometimes referred to as “knock-down” or “knock-out” depending upon whether or not the message is either partially or completely eliminated, allows researchers to determine the function of that gene.
One of the significant problems faced by developmental biologists, who do not work on an organism with well developed genetics and even for some who do, is how to inhibit the action of a gene of interest during its developmental process so as to learn about its normal biological functions. A widely accepted approach is to use the antisense technologies.
C. Cazenave, et al. in Nucleic Acids Res. 1989, 17, 4255 is directed to the first generation antisense oligonucleotides that were designed as short stretches of DNA (18-22mers) that form RNA-DNA hybrids with the target mRNA and act as substrates for RNase H to degrade the mRNA. The target mRNA is cleaved by RNase H and the fragments are subsequently broken down by nuclease activity. DNA oligonucleotides have had very limited applicability in developmental studies, both because they have non-specific toxic side effects and because degraded mRNAs are continually replaced by new transcription, making continued treatment with oligonucleotides a necessity.
Studies from a number of laboratories suggest that morpholino based oligonucleotides can be microinjected into zebrafish (Nasevicius, A. and Ekker, S. C. Nat. Genet. 2000, 26, 216), sea urchin (Eisen, J. S.; Smith, J. C. Development 2008, 135, 1735) or xenopus embryos (Coffman, J. A. et. al. Runx gene. BMC Biol. 2004, 2, 6), where they block gene expression and produce phenotypic effects during the early stage of embryogenesis. It is known in the art that light mediated activation of morpholino enables spatio-temporal regulation of ntl gene expression during zebrafish embryogenesis. The strengths of morpholinos as a tool for investigating vertebrate development is well described in a recent review by Ekker (Ekker, S. C. Morphants Yeast 2000, 17, 302). The greatest advantage is that phenotypes can be rapidly observed in animal models using a relatively inexpensive method. Ekker and colleagues also reported inhibition of several endogenous zebrafish gene and have compiled a database (The Morphant database) thereof detailing the phenotypes produced by morpholino oligonucleotides. Since then morpholinos have become superior agents for gene inhibition relative to other types of oligomers that might block translation, such as locked nucleic acid (LNA), peptide nucleic acid (PNA) or 2′-modified RNA.
Morpholino antisense oligomers taught in U.S. Pat. No. 5,185,444 and U.S. Pat. No. 5,034,506, are chemically modified nucleotides, widely used in gene regulation studies that cost about US$500-600 for 300 nanomol. The said morpholino-based oligomers block mRNA translation in binding 5′ UTR region, overcoming many of the limitations of regular DNA oligonucleotides.
The drawbacks in employing the above said morpholino based oligomers in developmental biology lies in the chiral nature of the morpholino phosphorodiamidate backbones that complicates their synthesis and purification. Further, adding an additional hurdle to the poor Morpholino transfection properties is the neutrality of its backbone comprising of phosphorodiamidate moiety that poses a serious limitation for use of these oligomers in tissue culture as well as in vivo studies. Accordingly, the delivery of morpholino into embryos requiring microinjection procedures, PNA (peptide nucleic acid) analogs constitute an alternative approach but they also have very limited applications due to their poor solubility in water under physiological conditions as taught in K. A. Urtishak, et. al., Dev. Dynamics; 228, 405, 2003.
Again to overcome the cellular transfection property of such morpholine based oligomers, M. H. Nelson, et al. in Bioconjug. Chem. 2005, 16, 959 and U.S. Pat. No. 7,468,418 illustrates the incorporation of cationic charge onto the morpholino oligomer through the conjugation with the arginine-rich peptide to study the effects on antisense activity and specificity. Although the insertion of cationic charge into the morpholino oligomers improved the antisense efficiency, however incorporation of arginine peptides for optimized results were found to be procedurally difficult and non-reproducible to the fullest extent. Furthermore, the structure of the morpholino oligomer of the above said prior art was not structurally modified wherein the product is a just a simple conjugate comprising a carrier peptide attached to a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence.
Reference is drawn to WO/2008/036127 that teaches a morpholino oligomer containing both uncharged and cationic intersubunit linkages. The oligomers are oligonucleotide analogs containing predetermined sequences of base-pairing moieties. The presence of the cationic intersubunit linkages in the oligomers, typically at a level of about 10-50% of total linkages, provide enhanced antisense activity, in various antisense applications, relative to the corresponding uncharged oligomers. Also provided are such oligomers conjugated to peptide transporter moieties, where the transporters are preferably composed of arginine subunits, or arginine dimers, alternating with neutral amino acid subunits. The said oligomers having an uncharged backbone, the cationic substitution takes place in the side chain associated with the phosphorodiamidate linkage, therefore the transfection efficiency is moderate and the product is costly because of the troublesome process of preparation.
J. Yuan, et al. in J. Virol. 2006, 80, 11510 illustrates peptide-conjugated morpholino oligomers targeting the internal ribosome entry site, in which morpholino have been conjugated to various cell-penetrating peptides (CPPs) in order to enhance both morpholino uptake into cells and antisense efficacy (M. H. Nelson, et al. Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity. Bioconjug. Chem. 2005, 16, 959). The conjugated morpholinos has a better binding ability to complimentary RNA in reduced length of 14 bases from 20 bases. Though Arginine peptide conjugated morpholino have demonstrated considerable efficacy against a number of RNA viruses including foot-and-mouth disease virus (A. Vagnozzi, et. al. in J. Virol. 2007, 81, 11669) in cell cultures and against Ebola virus, West Nile virus, murine corona viruses, and CVB3 in both cell culture and animal models, the limitations of such arginine rich peptide conjugated morpholino oligomers lies in its poor solubility in tissue culture media and complicated process of synthesis thereof.
It is thus clearly apparent from the above discussions that though modifications of the morpholino oligomers are known in the art, they suffer from serious drawbacks such as cumbersome synthetic procedures, poor solubility and reduced antisense efficacy.
Thus to address the need of the day there remains a long felt need in the art to bring about structural modifications in the above said morpholine oligomers through simple and cheap synthetic pathway to overcome the above said drawbacks by accomplishing improved antisense efficacy and increased solubility of such oligomers.